This research was supported by funding from NIH NCRR (R01 RR014166 to BHS), NIH NIDCD (R01 DC011422 BHS co-PI), the US Department of Agriculture (J Trumble PI; BH Smith co-PI) and Arizona State University. Funding for the workshop to train students (data in Figure 4) was provided by the science foundation of Chile.

<ol>
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The authors have no conflicts of interest to disclose.

This protocol presented two reliable methods for conditioning using the PER procedure. These are two of several methods that have been successfully employed27,21. We employ Method 2 for all experimental studies using PER as it is consistently reliable, even across different experimenters.
The same basic procedure has been adapted to many different kinds of studies with honey bees, including use of different conditioning stimuli and different behavioral reflexes. It has also been linked to investigations of the genetic basis of differences in learning28,29, the physiological correlates of olfactory perception and memory in the brain13,14,30, and the modulatory and molecular genetic bases of behavior15,16,31. Because of the advanced knowledge of the ecological relevance of olfactory learning to honey bees, which started with the first studies by Karl von Frisch over 100 years ago31, results from PER can be easily linked to survival needs of a colony. Most recently, it has been adapted to an agricultural study to investigate the sublethal behavioral effects of pesticides and environmental toxins17.
The basic procedure is powerful in that it can be applied to investigate problems in other species as well. Two moth species, Manduca sexta and Spodoptera littoralis, have been used in PER studies to investigate the neural basis of odor identification and each species&#8217; olfactory learning capabilities32,33. PER experiments with fruit flies have provided many insights into the molecular signaling cascades involved in chemosensation and learning34. And PER has recently been used to study habituation in the flea (Rhodnius prolixus)35, an important disease vector.
In general, the procedure is robust to changes in method; the use of different methods will likely produce the same relative difference between treatment groups. In spite of the relatively simple procedure, several problems can at times prevent honey bees from learning the odor-sucrose association. The following topics are possible alterations to the protocol and some of the more common problems that may arise during PER conditioning.
Considerations on Number and Type of Trials in the Conditioning Protocol
Any learning protocol will require exposure of the bees to a number of trials in the acquisition phase. This number depends largely on the difficulty of the task. Honey bees can learn a simple task after a single trial, but they need at least a three trials to induce long-term memory formation. As expected, the honey bees will require significantly more trials to adequately learn a difficult task. Generally, there is a maximum number of trials beyond which the bees no longer significantly improve their performance. This maximum depends on the specific task, the odor types and concentrations, and the sucrose concentration.
When using more than one type of trial with different odors, present the different trial types in a pseudorandom sequence to keep the bees from memorizing a simple sequence of odor presentations instead of learning the differences between the odors36. In these pseudorandom sequences, there must be an equal number of trials for each of the odor types. Also, the probability of a trial of one odor type being preceded by and followed by the same odor, or any of the other odors, must be equal for all the odors. For two odors &#8211; A and B &#8211; use the following sequence: ABBABAAB. Over eight trials each odor is presented four times. Concatenate identical sequences to reach the desired number of trials for each odor.
Regardless of the experimental design, there are a few parameters that must remain constant in order to optimize learning. The overlap between the CS and the US is critical for effective conditioning. The inter-trial interval (Figure 1, Table 1) needs to be constant and be optimized because irregularity or a too short or too long inter-trial interval can significantly influence the effectiveness of the conditioning protocol1.
Considerations on the Importance and Design of Behavioral Testing Trials
Responses recorded during the &#8216;acquisition phase&#8217; of an experiment, when the CS and US are presented together, can be useful. However, beware that the odor presentation conditions can differ from one type of trial to another. During reinforced trials, the odor followed by sucrose is presented in conjunction with a visual stimulus (movement of the device for delivering the sucrose droplet) that can affect the bee&#8217;s response. And the bees have only three seconds to show a response (four seconds of odor stimulation minus the one sec overlap with sucrose presentation; Figure 1). If the experiment involves the bees learning to differentiate two odors (e.g., reinforced and unreinforced), the unreinforced odor presented on alternate trials occurs without the visual stimulus of the reward presentation and the bees have the full four seconds to respond. Therefore, the responses to the two odors are not fully comparable during acquisition. With any conditioning protocol, do not rely solely on acquisition curves1. To better ascertain what bees have learned, introduce a series of unreinforced test trials, during which neither odor is reinforced, which ensures that testing of all stimuli takes place under identical conditions.
Depending on the specific purpose of the experiment, testing can consist of single test trials of the conditioned odor or a series of trials with the conditioned odor or a combination of the conditioned and novel odors. A single test trial of the conditioned odor provides a simple assay of whether the bees&#8217; remember the conditioned odor. However, the response to the first test trial may reflect the bees&#8217; motivation level as well as their recall of the conditioned odor. A series of test trials, either a series of extinction trials of the conditioned odor or a series of single tests of the conditioned and one or more novel odors, can also be used to assess memory. The series of extinction trials can assess how strongly the bees&#8217; remember the conditioned association. The stronger the recall the greater number of trials needed to extinguish the conditioned response. A series of single tests of conditioned and novel odors can also assess the specificity the bees&#8217; memory of the conditioned odor.
It is also imperative to condition and test both control and treated groups at the same time points. Comparing the performance of bees soon after conditioning to bees held for longer time periods is problematic because of the exposure to the sucrose US by feeding the bees to satiation. For example, a decrease in performance after long intervals could be due to memory decay or it could be due to changes in motivational state and/or learning induced by unreinforced exposure to the US, making the results ambiguous. Therefore, performance of a treatment group should always be judged relative to a control group conditioned and tested at the same time points.
Odor Concentration and Integrity
There are several ways in which the concentration and integrity of the odor (CS) can be compromised. The most prevalent problem with odor delivery is the depletion of odor cartridges from overuse (Figure 2). Replace the cartridges 10-12 uses (every 2 or 3 trials with groups of 5 honey bees) to avoid this pitfall. It is also critical to use fresh cartridges for test trials, since used cartridges may be differentially depleted and thus present unequal odor stimuli. Another common problem is odor cartridge contamination due to using the cartridge for more than one odor without completely cleaning it. A dirty or contaminated air flow can also unintentionally introduce additional olfactory stimulus (activated charcoal filters can prevent background contamination). This is especially problematic when coupling PER conditioning with measurements of the physiological responses to the odor. Leaky odor cartridges present a problem since the honey bees are exposed to odor before the trial begins, which reduces the salience of the odor stimulus. Loose fitting on the air supply for odor delivery can result in little or no odor delivery when the valve opens, artificially reducing the bees&#8217; response to the intended odor.
Sucrose Solution and Unconditioned Stimulus (Reward)
The amount, concentration, and integrity of the sucrose solution used as the US are vital to the success of the experiment. Conditioning is a function of the amount and concentration of sucrose-water solution used as the US37. The micrometer syringes used in Method 2 allow for very precise (down to 0.2 &#956;l) control of US delivery, and we recommend using them for both methods described. Use of the toothpicks (Method 1) is adequate for circumstances in which the expensive syringes cannot be used, such as, in training large groups of students, work in the field, or with lower budgets. Careful implementation of Method 1 is fine as long as timing is maintained with regular replacement of toothpicks to avoid buildup of sucrose (and the concentration delivered). However, using toothpicks it is more difficult to accurately control and estimate the amount and magnitude of the US delivered, which is important for conditioning studies1. The concentration of sucrose needed to provide a sufficient reward to keep the bees motivated to learn the conditioned association can depend on the difficulty of the task and the bees&#8217; internal state or time of year. The more difficult tasks require a higher sucrose solution for the bees&#8217; to successfully learn the task. Mold can buildup in sucrose solutions even at 5 &#176;C, which will compromise the integrity of the solution, affecting the bees&#8217; health and perception of the reward during the experiment. It is best to replace the solution every few days.
Precision, Timing, and Consistency of CS and US Delivery
The most critical issue about proper implementation of a PER procedure, or for that matter any conditioning procedure, involves precision, timing and consistency of CS and US delivery (Figure 1). Investigators who are new to the procedure frequently are imprecise about delivery of one or both stimuli. ISI&#8217;s that fail to allow for overlap the CS and US result in poor conditioning performance. The PLC automates an audible signal for the experimenter to deliver sucrose 3 sec after the onset of odor delivery. Investigators should hold the sucrose/water droplet close to the bee for rapid delivery after the signal. These procedures help entrain a consistent ISI. Placing a stopwatch by the conditioning arena allows for convenient time placement of the trial as well as monitoring the bees&#8217; time in the arena both before and after delivery of stimuli. That way the ITI&#8217;s can be relatively consistent and the entire procedure can be run at a controlled pace. ITI&#8217;s that are too short, for example less than 1 min, or too long can lead to poor conditioning performance1.
Seasonal, environmental, and Contextual Effects on Honey Bee Performance
Honey bees&#8217; performance can be significantly influenced by its surroundings both prior to and during conditioning. Frequently, the fluctuations in temperature and food availability that come with the changing seasons will alter the bees&#8217; motivation to learn. When flowers are in bloom, the bees&#8217; motivation to learn the conditioned association in the lab decreases38. When the colony is stressed &#8211; from extreme temperatures, food shortages, or disease-related stress &#8211; the bees&#8217; will show a reduction in their learning performance inside. Honey bees kept in a flight room may learn well for a while, but there too the stress of disease and aging degrade their learning performance over time. The context during conditioning can also decrease the bees&#8217; performance. Any extraneous odors, movement, and other stimuli can distract the bee from the experimental stimuli. To avoid this problem, maintain a reasonably consistent, simplified visual context.
Genotype and Experience Affects PER Performance
Honey bee workers can differ considerably in performance on any conditioning procedure due to task specialization, genotype, or other environmental factors20. Therefore, it is important to standardize, as much as possible, the types of animals used in an experiment in order to reduce inter-individual variation. In a colony led by an open-mated queen, which means she mated with many different drones, the workers will differ in paternal genotype. Genetic background can lead to dramatic differences in sensory responsiveness39 and learning performance29. Using colonies led by queens instrumentally inseminated by sperm from single drones28 reduces this inter-individual variation.
The protocols described above, includes a method for collecting honey bee workers from the nest entrance. However, these honey bees differ from each other in regard to age or behavioral task. They may be young (inexperienced) or older (more experienced) foragers. They may be young honey bees making their first orientation flights. Or they may be guard bees. To reduce the variability, mark the bees with a quick-drying enamel paint or marking tags either as they emerge as adults (to control for age) and/or as they begin foraging (to control for experience). Then, a few days later, collect the marked bees for conditioning. Workers that are engaged in nursing behavior can be collected from the frames within the hive. Nurses can be positively identified when they insert their heads into a brood cell to feed and care for the larva inside.
Using Virgin Queens or Drones for PER Conditioning
In addition to worker honey bees, virgin honey bee queens and drones can be readily conditioned in a PER procedure for studies aimed at developing genetic lines of honey bees that differ in learning performance28. Virgin queens should be collected soon after they emerge from pupation and placed directly into restraining harnesses without anesthetization. Young, immature drones collected from the brood comb inside the colony are generally not motivated to learn. After they have begun mating flights, drones readily learn PER tasks28. They should be collected as they return from a mating flight and maintained overnight in a small cage in a colony used for rearing queens. Do not attempt to harness them the day before conditioning; they do not survive well in harnesses overnight. A couple of hours prior to conditioning the drones can be collected from the cages and placed into the restraining harnesses without anesthetization.
Conclusions
This PER procedure, in the way of methods, amounts to a starting point for designing PER experiments. Most PER protocols will require that the procedure outlined be changed in some way to implement the specific goals of the experiment and accommodate multiple treatment groups. It is easy to implement. However, proper implementation requires attention to detail and practice. Once mastered it can be a powerful procedure to add as a research tool for several basic and applied research programs with different insect species.

Many insects learn about ecologically relevant stimuli, and they then change their behavioral responses to those stimuli in order to adapt to new predictive relationships in their environment. Several different mechanisms can underlie this behavioral plasticity (e.g., nonassociative, associative/Pavlovian, and operant/instrumental1). These types of plasticity differ in how the stimuli or behaviors are associated with important events, such as the occurrence of food, a mate, or danger. Understanding these forms of plasticity is very important for basic research into how the nervous system changes to encode new memories2. It is also important for understanding the adaptive behaviors of insects that are important vectors of disease (e.g., tsetse and mosquito) and insects that are agriculturally important, either in crop production (honey bees) or as pests.
Studying behavioral plasticity in any animal requires a level of experimental control over a number of variables that is not achievable in the field1. It requires the development of a robust conditioning protocol that can be employed under more controlled conditions, yet which are still relevant to behavior under natural conditions. The honey bee (Apis mellifera) is an excellent model for how to develop a protocol for performing controlled analyses of behavioral plasticity3,4. The Proboscis Extension Response (PER) in honey bees is a natural behavioral reflex in which the honey bee extends its proboscis in response to antennal stimulation with a sugar solution. During normal foraging behavior, PER occurs when the honey bee finds nectar in a flower. Fortunately, honey bees will readily exhibit this simple and easily quantifiable behavior in the laboratory. This makes it possible to study, in a controlled setting, the mechanisms that influence this ecologically relevant behavior5. PER can also be used within a conditioning protocol to investigate stimulus perception and learning and memory under different treatment conditions, which are designed to reveal the behavioral and neural mechanisms that underlie the plasticity6.
Since the first studies by Kuwabara7, PER conditioning has been widely used to reveal nonassociative, associative and operant mechanisms that underlie behavioral plasticity in honey bees8. These mechanisms are identical to those revealed in studies of freely flying honey bees9. Unlike studies of freely flying honey bees, PER conditioning protocols can be coupled with electrophysiology10,11 or live-cell fluorescence imaging12-14 of the brain. Furthermore, PER protocols allow for experimental manipulation of neural pathways via pharmacological or molecular genetic treatments to test hypotheses about the roles of specific components of the network, such as neuromodulators15,16. PER protocols have also provided an important way to evaluate the sublethal effects of environmental conditions as well as toxins on health and foraging efficiency of honey bees17.
This procedure describes two odor delivery methods in parallel. Method 1 is a version of the odor and unconditioned stimulus (sucrose) delivery that provides an inexpensive and technically simple method for presenting the odor and sucrose reward. This method is good for basic training and when automation is not possible. It is an excellent way to introduce this technique to a classroom or teaching laboratory. During conditioning for experiments involving more difficult tasks and coupled physiological assessments of odor perception, learning, or memory, it is very important to accurately and precisely regulate the onset, duration delivery timing of stimuli. For the most reliable stimulus delivery, use a means of automating the odor delivery and a precise method of reward delivery. Method 2 employs automated odor delivery and more precise sucrose delivery. It is technically more sophisticated and requires more for the initial setup than Method 1, but it significantly increases the consistency of the timing and quantity of stimuli used for conditioning and should be used whenever possible.

<table border="0" cellpadding="0" cellspacing="0" width="1278">
	<tbody>
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			<td height="21" style="height:21px;width:371px;">Name of Material/ Equipment</td>
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			<td style="width:587px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Sucrose</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9378-1KG</td>
			<td style="width:587px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Odorant compounds (for example):</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td rowspan="6" style="width:587px;">For additional examples of odorants, see any of the papers on olfactory processing from the Smith lab</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">1-hexanol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">471402-100ML</td>
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			<td height="21" style="height:21px;width:371px;">nonanal</td>
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			<td style="width:119px;">131210-100ML</td>
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			<td height="21" style="height:21px;width:371px;">Hexane</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">296090-1L</td>
			<td style="width:587px;"></td>
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			<td height="21" style="height:21px;width:371px;">Heavy Mineral Oil</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">330760-1L</td>
			<td style="width:587px;">Make sure it&#8217;s odorless</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Ethanol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">459836-1L</td>
			<td style="width:587px;"></td>
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			<td height="21" style="height:21px;width:371px;">Scintillation vials</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z190535-1PAK</td>
			<td style="width:587px;">Use a small drill bit to bore a small hole in the cap of the vials&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bee harness</td>
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			<td style="width:587px;">Custom-made from 0.9 cm diameter plastic soda straws or hard plastic/metal tubing</td>
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			<td height="21" style="height:21px;width:371px;">Duct tape</td>
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			<td height="21" style="height:21px;width:371px;">Kimwipes</td>
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			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z560847-3EA</td>
			<td style="width:587px;">For the 70% ethanol&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Small fan in mountable housing</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td style="width:587px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Dryer tubing</td>
			<td style="width:203px;"></td>
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			<td style="width:587px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
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			<td style="width:587px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">FOR METHOD 1 ONLY</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name</td>
			<td style="width:203px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:587px;">Comments</td>
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			<td height="21" style="height:21px;width:371px;">20 ml disposable plastic syringes</td>
			<td style="width:203px;">Cole-Parmer</td>
			<td style="width:119px;">WU-07945-18</td>
			<td style="width:587px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">15 mm filter paper circles</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z274844-1PAK</td>
			<td style="width:587px;"></td>
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			<td height="21" style="height:21px;width:371px;">Pushpins</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td style="width:587px;"></td>
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			<td height="21" style="height:21px;width:371px;">Toothpicks</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
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		<tr height="21">
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			<td style="width:203px;"></td>
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			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:203px;"></td>
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			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">FOR METHOD 2 ONLY</td>
			<td style="width:203px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name</td>
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			<td height="21" style="height:21px;width:371px;">Gilmont Micrometer syringe, 0.2 mL</td>
			<td style="width:203px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07840-00</td>
			<td style="width:587px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Gilmont micrometer syringe tip</td>
			<td style="width:203px;">Cole-Parmer</td>
			<td style="width:119px;">EW-07841-00</td>
			<td style="width:587px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">26G 3/8&#8221; Leur hub hypodermic needles</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-10</td>
			<td style="width:587px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:371px;">1cc tuberculin syringes (plastic/glass)</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z181641-1EA&#160;&#160; OR &#160;&#160;Z192090-200EA</td>
			<td style="width:587px;">glass tuberculin syringes are available, but plastic syringes are much less expensive and will work well for a limited number of uses</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Small rubber/silicone restrictors</td>
			<td>Cole-Parmer</td>
			<td>EW-95702-02</td>
			<td>Made from 4.8 mm outer diameter silicone tubing or the rubber tips of the 1 cc syringe plungers</td>
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			<td height="21" style="height:21px;width:371px;">Parafilm</td>
			<td style="width:203px;">Sigma-Aldrich</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:371px;">75 X 100 mm filter paper</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z695106-500EA</td>
			<td style="width:587px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Direct Logic 05 Programmable Logic Controller</td>
			<td style="width:203px;">Koyo Electronics Industries Co, Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:587px;">http://www.koyoele.co.jp/&#160;&#160;&#160;&#160;&#160;&#160;&#160; OR&#160;&#160;&#160;&#160;&#160;&#160; english/support/dlc.html#plc</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:371px;">1 mm, 4mm &#38; 6 mm inner diameter PVC or silicone tubing&#160;</td>
			<td style="width:203px;">Cole-Parmer</td>
			<td style="width:119px;">Various</td>
			<td style="width:587px;">Cole-Parmer has a wide selection of suitable tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Polypropylene connectors &#38; leur fittings</td>
			<td style="width:203px;">Cole-Parmer</td>
			<td style="width:119px;">Various</td>
			<td style="width:587px;">Cole-Parmer has a wide selection of connectors and fittings for many tube sizes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">65-mm Correlated Flowmeter</td>
			<td style="width:203px;">Cole-Parmer</td>
			<td style="width:119px;">EW-03216-08</td>
			<td style="width:587px;">Aluminum with Glass float; For Liquids and Gases, With Valve</td>
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			<td height="21" style="height:21px;width:371px;">&#160;&#160;&#160;&#160; OR&#160;</td>
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			<td height="42" style="height:42px;width:371px;">Tetra Whisper 300 (Tetratek DW96-2) Aquarium Air Pump</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:371px;">LIF series Solenoid Valves for .042 &#34; ID Tubing, Configuration &#34;E&#34;</td>
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			<td height="21" style="height:21px;width:371px;">PC-Board 12VDC 70dB Piezo Buzzer</td>
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			<td style="width:119px;">273-074</td>
			<td style="width:587px;">www.radioshack.com</td>
		</tr>
	</tbody>
</table>

a proboscis extension response protocol for investigating behavioral plasticity in insects: application to basic, biomedical, and agricultural research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

We present two examples of use of the PER protocols described above. The first example made use of Method 2 to study how honey bees perceive different odors as a function of the molecular similarity to odor used as the CS5,24,25. The second is an example of the use of Method 1 and some of the precautions that must be taken when beginning to use PER conditioning experiments in the research laboratory.
Studies such as this one have been used to describe the olfactory &#8216;perceptual space&#8217; of honey bees and moths in conjunction with bioimaging13 or electrophysiological analyses26. Honey bees (n = 20) were conditioned to associate the odorant decanal with sucrose reinforcement over 12 forward pairing conditioning trials (Figure 3A). Approximately 10% of the honey bees responded &#8216;spontaneously&#8217; on the first trial, which is normal. After that the percentage that responded increase over the next few trials until 100% of the honey bees responded on the fifth trial and every subsequent trial. Several studies have shown that this increase in response is specific to the forward pairing condition (Figure 1) relative to several important control procedures3. After the acquisition phase, each honey bee was subjected to a series of unreinforced test trials (Figure 3B), which involved exposure to the odor CS and to several other odorants that systematically differed from the CS in molecular structures. Honey bees responded most strongly to the CS. Their responses to the other odorants decreased as a function of the systematic changes in structure, with the lowest responses to the odorants least like the CS.
In Figure 4, the results from a recent workshop in which students learned, for the first time, how to condition honey bees using Method 1 are illustrated. Students first conditioned honey bees by forward pairing methycyclohexanone (MCH) or octanol (OCT) with sucrose reinforcement (Figure 4A). Percent responses were lower on the first trial and increased on subsequent trials, indicating that the bees learned the odor-sucrose association. There were differences in the degree that the bees learned the conditioned association across the different groups of students. In our experience, these kinds of differences could be due to the odor that was used as the CS, to the imprecise elements in the method (Method 1), or to the training level of the experimenters. In the third case, performance quickly becomes more consistent with the experimenters&#8217; increased experience. After conditioning, each honey bee was tested twice with each odor. In groups of honey bees that had been conditioned with forward pairing of OCT or MCH, the response was, as expected, strongest to the conditioned odor (Figure 4B).
The second group of honey bees that experienced forward pairing to OCT was conditioned in parallel to another group in which the odor OCT was backward paired with sucrose. Backward pairing is one of several types of control procedures to demonstrate that the increase in response is specific to forward pairing and not due to another process, such as non-specific arousal from sensitization. For backward pairing, the sucrose US and odor CS are presented in reverse order to what is shown in Figure 1. Acquisition data from backward pairing are not shown because the CR cannot be scored given the presentation of the odor CS after presentation of sucrose. As would be predicted for associative conditioning, the response levels to OCT were higher in the forward paired group relative to the backward group (Figure 4C).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51057/51057fig1highres.jpg" width="500" /> 
Figure 1. Diagrams of experimental design for conditioning using forward pairing. See Table 1 for definitions of the terms used in this figure. (A) The CS (odor) precedes and overlaps with the US (sucrose/water solution). The relationship between the CS and US shown is optimal for conditioning honey bees and moths. But the optimal ISI can depend on the conditioning protocol and animal species1. (B) Breakdown and relative timing of CS, CR, US, and UR. The CR that occurs either before US presentation or during unreinforced &#8216;test&#8217; trials is the dependent measure for experimental protocols.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51057/51057fig2highres.jpg" width="500" /> 
Figure 2. Odor depletion with reuse of cartridges. For these data, odor cartridges were set-up with hexanol as described in the text. Hexanol is commonly used as a CS in PER studies; however, the actual rate of depletion will depend on the odor and solvent. Cartridges were then used once for 4 sec every minute over 8 trials (assuming 5 honey bees/trial in a standard experiment that would equal 40 uses). The dilutions in the indicated solvent, which were relatively standard for PER experiments, were 2.0 M (A) and 0.2 M (B). (A, B)The odor was sampled after the first and the eighth trials by adsorption onto a Solid Phase Microextraction (SPME) fiber that was then desorbed onto a gas chromatograph. The relative areas under the peaks were higher after the first trial relative to the eighth for both solvents and for both concentrations (Wilcoxon matched pairs signed rank test p &#60; 0.05 [*] or greater), which shows that less odor was delivered from the cartridge after the cartridge had been used a number of times. (C, D) The same data showing just the eighth trial with the y-axis expanded. It shows that depletion was slightly greater for hexane relative to mineral oil, but only at the lower concentration.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51057/51057fig3highres.jpg" width="500" /> 
Figure 3. Acquisition and test using decanal as the CS and method 2 to conditioning. (A) Trial-by-trial response (CR) over 12 forward pairing trials (Figure 1). Data are from n = 20 honey bees conditioned in four groups of 5 honey bees each. The ISI was 3 sec and the ITI was 6 min. (B) Unreinforced tests using odors that differ from decanal by carbon chain length and/or position of the carbonyl carbon. The odors were presented in a randomized series across the four groups and were interspersed with reinforced trials with decanal (every two to three trials) in order to avoid extinction of the CR.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51057/51057fig4highres.jpg" width="500" /> 
Figure 4. Data from a workshop during which graduate students were trained to condition honey bees using PER method 1. (A) Acquisition data from three groups of honey bees conditioned to octanol (n = 23 and 15 for Gps 1 and 2, respectively) and methylcyclohexanone (n = 10). (B) CRs during unreinforced tests with both odors; four trials presented in random series across groups. (C) Unreinforced tests with both odors in two groups of honey bees, one forward paired (Gp 1, n = 23) and one backward paired (n = 25).
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Term (abbreviation)</td>
			<td>Definition</td>
			<td>Relevant Example</td>
		</tr>
		<tr>
			<td>Conditioned Stimulus (CS)</td>
			<td>A stimulus that elicits little or no response at first and signals US</td>
			<td>ODOR</td>
		</tr>
		<tr>
			<td>Conditioned Response (CR)a</td>
			<td>The response to the CS after association with the US</td>
			<td>PROBOSCIS EXTENSION</td>
		</tr>
		<tr>
			<td>Unconditioned stimulus (US)</td>
			<td>A biologically significant stimulus that elicits a response</td>
			<td>SUCROSE/WATER SOLUTION</td>
		</tr>
		<tr>
			<td>Unconditioned Response (UR)a</td>
			<td>The response to the US</td>
			<td>PROBOSCIS EXTENSION</td>
		</tr>
		<tr>
			<td>Inter-Stimulus Interval (ISI)</td>
			<td>The time between onset of the CS and onset of the US</td>
			<td>ODOR to SUCROSE interval</td>
		</tr>
		<tr>
			<td>Inter-Trial Interval (ITI)</td>
			<td>The time between successive CS-US pairings for a single animal</td>
			<td>ODOR (trial n) to ODOR (trial n+1) interval</td>
		</tr>
	</tbody>
</table>
Table 1. Important Terminology. Refer to Figure 1 for an illustration of these terms. aFor PER conditioning the CR and UR are the same, although for other types of associations and reflexes the CR and UR may differ.
<a href="/files/ftp_upload/51057/BEE_4.avi" target="_blank">Movie 1.</a> A video of a honey bee performing PER after it had been conditioned to respond to an odor. Note the PER response of the bee approximately 1.5 sec following odor onset. Tracking software mapped the antennal movements in response to presentation of the odor. The lines plotted in the upper right hand corner of the video depict the actual antennal movements. The two graphs at the bottom of the screen show antennal movements on the X (left) and Y (right) coordinates. The vertical lines in the graph shown in the video indicate, left to right, light ON, PER, light off and proboscis retraction.

Watch this Scientific Journal Video about A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research at JoV

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

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33.3K Views.  Arizona State University. The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species. 

Video: A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research